In the central nervous system (CMS), the extracellular concentration of the excitatory amino acid L- glutamate must be tightly regulated to ensure proper neural signaling. This is achieved mainly by excitatory amino acid transporters (EAATs) located in the plasma membranes of both glial cells and neurons. Many neurodegenerative diseases such as Parkinson's, Alzheimer's, cerebral ischemia, epilepsy, and amyotrophic lateral sclerosis (ALS) are linked to abnormal glutamate homeostasis. Previous efforts to treat these diseases focused on glutamate receptors, rather than glutamate transporter proteins, and have yielded little success. This suggests the need for alternative approaches to treat the large number of neuropathological conditions associated with abnormal glutamate homeostasis. To find novel approaches for treating these various diseases, we have focused our research on elucidating the biophysical properties of glutamate transporters. This is a credible alternative based on the broad spectrum of drugs which target various neurotransmitter transporter proteins, including drugs used to treat depression, anxiety, obesity, and epilepsy. Recent publication of bacterial glutamate transporter crystal structures has provided tantalizing clues as to how these transporters function. We aim to experimentally test predictions made from analyzing the crystal structures in order to define the precise glutamate transporter gating mechanism. This information will provide insight toward the development of novel compounds with clinical applications for the treatment of neurodegenerative disorders. To conduct our experiments, mRNA transcribed from cDNA encoding wild-type (WT) and mutated versions of the human glutamate transporter EAATS will be injected into Xenopus oocytes. 3-6 days later, glutamate-induced membrane currents will be recorded by two- electrode voltage clamp (TEVC). The mutated versions of EAATS will be made using site-directed mutagenesis to introduce cysteine residues which can react with methanethiosulfonate reagents. We aim to investigate the hypothesis that the extracellular and putative intracellular gates undergo conformational changes subsequent to the binding of glutamate and that these conformational changes are important for the translocation step of the glutamate transport cycle. We will also determine the pore-like region of the glutamate transporter that functions to provide a pathway for glutamate uptake. All of our experiments will utilize the technique of MTS accessibility under conditions that favor different states of the glutamate transporter cycle. These experiments will allow us to define the glutamate transporter gating mechanism and will lead to new avenues of research to treat neurodegenerative disease.